U.S. patent application number 12/281130 was filed with the patent office on 2009-10-01 for g-csf liquid formulation.
Invention is credited to Michael Mack.
Application Number | 20090247450 12/281130 |
Document ID | / |
Family ID | 38226349 |
Filed Date | 2009-10-01 |
United States Patent
Application |
20090247450 |
Kind Code |
A1 |
Mack; Michael |
October 1, 2009 |
G-CSF LIQUID FORMULATION
Abstract
The invention relates to G-CSF liquid formulations that have a
long shelf-life and to methods for producing the same. The
invention also relates to liquid formulations containing G-CSF as
the active substance, acetate as the buffer substance, polysorbate
20 or polysorbate 80 as the surfactant and optionally
pharmaceutically acceptable adjuvants, the formulations having a pH
between 4.1 and 4.4.
Inventors: |
Mack; Michael; (Bad Vilbel,
DE) |
Correspondence
Address: |
KNOBBE MARTENS OLSON & BEAR LLP
2040 MAIN STREET, FOURTEENTH FLOOR
IRVINE
CA
92614
US
|
Family ID: |
38226349 |
Appl. No.: |
12/281130 |
Filed: |
March 1, 2007 |
PCT Filed: |
March 1, 2007 |
PCT NO: |
PCT/EP2007/051947 |
371 Date: |
April 6, 2009 |
Current U.S.
Class: |
514/1.1 |
Current CPC
Class: |
A61K 38/193 20130101;
A61P 43/00 20180101; A61K 9/0019 20130101 |
Class at
Publication: |
514/8 |
International
Class: |
A61K 38/18 20060101
A61K038/18; A61P 43/00 20060101 A61P043/00 |
Foreign Application Data
Date |
Code |
Application Number |
Mar 1, 2006 |
DE |
10 2006 009 437.9 |
Claims
1. A liquid formulation of G-CSF, comprising G-CSF as an active
ingredient, acetate as a buffer, Polysorbate 20 and/or Polysorbate
80 as a surfactant and optionally pharmaceutically acceptable
excipients, wherein the formulation has a pH value in the range of
between 4.1 to 4.4.
2. The liquid formulation of claim 1, wherein the pH value is
between 4.1 to 4.3.
3. The liquid formulation of claim 2, wherein the pH value is
between 4.2 to 4.3.
4. The liquid formulation of claim 3, wherein the pH value is
4.25.
5. The liquid formulation of claim 1, wherein the concentration of
acetate buffer is between 2 and 50 mmol/l.
6. The liquid formulation of claim 5, wherein the concentration of
acetate buffer is 10 mmol/l.
7. The liquid formulation of claim 1, wherein the formulation
comprises sorbitol and/or mannitol as a pharmaceutically acceptable
excipient.
8. The liquid formulation of claim 7, wherein the formulation
contains comprises sorbitol.
9. The liquid formulation of claim 1, wherein the formulation does
not contain a preservative.
10. The liquid formulation of claim 1, wherein the formulation does
not contain amino acids.
11. The liquid formulation of claim 1, wherein the formulation does
not contain polymeric stabilizing agents.
12. The liquid formulation of claim 1, wherein the pH value is
adjusted with NaOH.
13. The liquid formulation of claim 1, further comprising sodium
ions.
14. The liquid formulation of claim 1, consisting essentially of
G-CSF, Polysorbate 20 and/or Polysorbate 80, sorbitol, acetate as a
buffer and sodium.
15. The liquid formulation of claim 1, wherein the surfactant is
Polysorbate 20.
16. A method for producing the liquid formulation of G-CSF
according to claim 1, comprising mixing the G-CSF with a solution
comprising the acetate buffer, the Polysorbate 20 and/or
Polysorbate 80 surfactant and the optionally pharmaceutically
acceptable excipients.
Description
[0001] The present invention relates to storage-stable liquid
formulations of G-CSF as well as to methods for their production.
In particular, the present invention relates to liquid formulations
containing G-CSF as active agent, acetate as buffer substance,
polysorbate 20 or polysorbate 80 as tenside and optionally
pharmaceutically acceptable excipients, wherein the formulations
have a pH value in the range of between 4.1 to 4.4.
[0002] G-CSF (granulocyte colony stimulating factor) is a naturally
occurring growth factor belonging, in a broader sense, to the
family of cytokines and, more precisely, to the group of colony
stimulating factors. G-CSF plays a decisive role in hematopoiesis
and enhances the proliferation and differentiation of hematopoietic
precursor cells and the activation of neutrophils. It is due to
said characteristics that G-CSF is applied in various medical
areas, like for example in the reconstitution of normal blood cell
populations subsequent to chemotherapy or irradiation or for
stimulating the immune response towards infectious pathogens. In
clinical practice, G-CSF is thus mainly applied in tumor prevention
and, in particular, in the treatment of neutropenia as a
consequence of chemotherapy and furthermore in the course of bone
marrow transplantations and in the treatment of infectious
diseases.
[0003] The recombinant production of G-CSF was first described in
patent literature in WO-A-87/01132 in 1987. The first commercial
G-CSF preparation on the basis of recombinant G-CSF was approved in
Germany in 1991 and is produced and distributed by Amgen under the
trade name Neupogen.RTM..
[0004] According to the German physicians desk reference ROTE LISTE
2005, the product Neupogen.RTM. (prefilled syringes) consists of
the following components: G-CSF at a concentration of 600 .mu.g/ml
or 960 .mu.g/ml, sodium acetate, sorbitol, polysorbate 80 and
water.
[0005] In the art, various patent documents deal with
pharmaceutical preparations of G-CSF. In EP-A-0 373 679,
formulations of G-CSF are described, in which the protein is
stabilized by the presence of an acid, an acidic pH value and a low
conductivity of the formulation.
[0006] DE-A-37 23 781 relates generally to the use of G-CSF in
combination with a pharmaceutically acceptable surface-active
agent, saccharide, protein or a pharmaceutically acceptable high
molecular compound.
[0007] In WO-A-94/14465, the use of maltose, cellobiose,
gentiobiose, isomaltose, raffinose, sucrose and further sugars for
stabilizing preparations containing G-CSF is described.
[0008] In WO-A-94/14466, preparations containing G-CSF are
disclosed which contain an amount of tenside that is smaller than
the amount of G-CSF employed, as well as a buffer substance.
[0009] WO-A-93/03744 describes formulations containing G-CSF which
contain a preservative that is chlorobutanol, benzyl alcohol, or
benzalkonium.
[0010] EP-A-0 988 861 discloses formulations containing G-CSF which
contain HEPES, TES, or tricine as buffer substance.
[0011] WO-A1-2005/042024 discloses pharmaceutical preparations of
G-CSF having a pH value of more than 4.0 which contain an acid and
are free of tensides.
[0012] It is the problem underlying the present invention to
produce a G-CSF preparation that can be stored for longer time
periods in liquid form and that does not require stabilizing
additives like HSA, amino acids or preservatives. In this way, a
storage-stable G-CSF liquid formulation is to be provided that
preferably avoids any risk with respect to the tolerability of the
formulation.
[0013] According to the present invention, this problem is solved
by means of the subject of claim 1. Preferred embodiments are
defined in the dependent claims.
[0014] It was found that, even in the absence of HSA, amino acids,
or polymeric stabilizers, liquid G-CSF compositions containing
acetate as buffer substance and polysorbate 20 and/or polysorbate
80 as tenside and having a pH value in the range of between 4.1 to
4.4 can be stored in liquid form for longer time periods without
significant losses in stability.
[0015] Thus, the present invention relates to a storage-stable
aqueous liquid formulation of G-CSF comprising, besides recombinant
human G-CSF as active agent, acetate as buffer and polysorbate 20
(polyoxyethylene sorbitan monolaurate, also referred to as Tween
20) or polysorbate 80 (polyoxyethylene sorbitan monooleate, also
referred to as Tween 80) or a mixture thereof as tenside and having
a pH value in the range of between 4.1 to 4.4.
[0016] In a preferred embodiment, the tenside is polysorbate
20.
[0017] However, it was also found that further polyoxyethylene
sorbitan alkyl esters are also suitable for the use as detergent in
the formulations of the present invention.
[0018] In general, the tenside is contained at a concentration in
the range of between 0.0005% (w/v) to 0.05% (w/v), preferably in
the range of between 0.001% (w/v) to 0.01%, particularly preferably
in the range of between 0.002% (w/v) to 0.008%, and most preferably
in the range of between 0.004% (w/v) to 0.006% (w/v), in relation
to the total volume of the solution. Normally, the formulations
according to the present invention contain the tenside polysorbate
20 and/or 80 at a concentration of 0.004% (w/v), 0.005% (w/v), or
0.006% (w/v).
[0019] The concentration of the buffer substance acetate is
preferably selected in such a way that, with the pH value in the
range of between 4.1 to 4.4 according to the present invention,
both the pH-stabilizing effect and a sufficient buffer capacity are
achieved, while simultaneously the ionic concentration and thus the
conductivity are kept as low as possible in order to avoid
aggregate formation. In general, acetate is contained at a
concentration in the range of between 0.5 to 150 mmol/l, preferably
in the range of between 1 to 100 mmol/l, particularly preferably in
the range of between 2 to 50 mmol/l, and most preferably between 5
and 20 mmol/l. Normally, the concentration of the acetate is 10
mmol/l.
[0020] The buffer substance acetate can be employed both in form of
the free acid and in form of the salt. As salts, in particular the
physiologically acceptable salts are employed, for example alkali
or ammonium salts, preferably the sodium salt.
[0021] The pH value of the formulation lies within a range from
more than 4.0 to 4.5, in particular between 4.1 and 4.4, preferably
between 4.1 and 4.3 or between 4.15 and 4.35, particularly
preferably between 4.2 and 4.3 or between 4.25 and 4.35, also
particularly preferably between 4.25 and 4.3, and most preferably
at about 4.25 or about 4.3.
[0022] If desired, the pH value of the composition can additionally
be adjusted to a pH value of more than 4.0 to 4.5, from 4.1 to 4.4,
from 4.1 to 4.3, from 4.15 to 4.35, from 4.2 to 4.3 or from 4.25 to
4.35, from 4.25 to 4.3 or to about 4.25 or about 4.3 with the aid
of further acids or bases. Suitable acids are, for example,
hydrochloric acid, phosphoric acid, citric acid and sodium or
potassium hydrogen phosphate. Suitable bases are, for example,
alkali and alkaline earth hydroxides, alkali carbonates, alkali
acetates, alkali citrates and dialkali hydrogen phosphates, for
example sodium hydroxide, sodium acetate, sodium carbonate, sodium
citrate, disodium and dipotassium hydrogen phosphate and
ammonia.
[0023] Preferably, the pH value is adjusted by means of NaOH. In
one embodiment, as a consequence of adjusting the pH value with
NaOH, the formulation of the present invention thus further
contains sodium ions.
[0024] In one embodiment, the formulation further contains a polyol
as pharmaceutically acceptable additive, in particular a sugar
alcohol, which particularly preferably is mannitol or sorbitol.
Said additives are particularly suitable as isotonizing agents for
making the compositions of the present invention isotonic with the
patient's blood.
[0025] Normally, the concentration of the polyol is up to 10.0%
(w/v) in relation to the total volume of the composition.
Preferably, the concentration is up to 8.0% (w/v), particularly
preferably up to 6.0% (w/v). Particularly preferably, sorbitol or
mannitol is contained at a concentration of 5.0% (w/v).
[0026] The concentration of G-CSF depends on the respectively
desired concentration of the agent in the prefilled syringe, in
which the liquid formulation according to the present invention is
stored and eventually applied. The trade product Neupogen.RTM.
(ROTE LISTE 2005, No. 51 038) is available, for example, in the
following concentrations: 300 .mu.g/0.5 ml; 480 .mu.g/0.5 ml and
300 .mu.g/1.0 ml. Herein, 10 .mu.g protein correspond to about 1.0
million International Units (IU). In the case of the trade product
Granocyte (ROTE LISTE No. 51 036), the activity of the recombinant
G-CSF is slightly higher; here, 10 .mu.g protein correspond to an
activity of 1.28 million IU.
[0027] Within the scope of the present invention, typical G-CSF
concentrations are between 0.01 and 3.0 mg/ml, preferably between
0.1 and 2.5 mg/ml, particularly preferably in a range of between
0.5 and 2.0 mg/ml, and most preferably between 0.6 and 1.5 mg/ml.
The concentrations 0.6 mg/ml and 0.96 mg/ml represent preferred
embodiments. Here, the activity of the employed G-CSF normally is
about 1.0.+-.0.6.times.10.sup.8 units/mg.
[0028] In more highly concentrated starting solutions, often
referred to as bulk solutions, the agent concentration can also be
higher; potentially 5 .mu.g/ml and more.
[0029] The compositions according to the present invention can
contain further conventional, in particular physiologically
acceptable, stabilizers and/or excipients or additives. For example
further tensides, isotonizing agents, reducing agents,
antioxidants, complexing agents, cosolvents, diluents and
chaotropic agents.
[0030] It is, however, preferred that the formulation does not
contain any polymeric stabilizers. Thus, for example polyalkylene
glycols like polyethylene glycol, hydroxyethyl starch, dextrans,
cyclodextrins, but also proteins like HSA and other plasma proteins
or gelatin are to be omitted.
[0031] For injection purposes, the use of pure water is preferred.
However, further conventional solvents that are suitable for
pharmaceutical preparations can also be employed. Solvents like
glycerol, polyethylene glycol and propylene glycol are preferably
omitted, however.
[0032] The employment of buffer substances like tartrate,
succinate, HEPES, TES and tricine is also omitted.
[0033] Preferably, amino acid stabilizers are also omitted.
[0034] On the whole, it is preferred to keep the number of
different excipients in the formulation as low as possible.
Correspondingly, sugars other than mannitol or sorbitol are
preferably avoided and the buffer substances contained in the
formulation are preferably narrowed down to acetate
exclusively.
[0035] The components of the formulation can be obtained from
conventional sources, for example from the company Sigma or the
company Merck.
[0036] The recombinant G-CSF can be produced and purified according
to state-of-the-art protocols.
[0037] The G-CSF is biologically active G-CSF that is capable of
enhancing the differentiation and proliferation of hematopoietic
precursor cells and of effecting the activation of mature cells of
the hematopoietic system. Thus, the G-CSF formulation is suitable
for treating indications in cases where the administration of G-CSF
is advantageous. It is understood that the term "biologically
active human G-CSF" also includes mutants and modifications of
G-CSF, whose amino acid sequence is altered as compared to the wild
type sequence, but which have a biological activity similar to that
of the wild type G-CSF, like for example those described in WO
01/87925 and EP 0 456 200. G-CSF in the sense of the present
invention also denotes G-CSF conjugates in which the protein is
present in conjugated form, for example with polymers like for
example polyalkylene glycols, in particular with polyethylene
glycol, as so-called PEGylated G-CSF or PEG-G-CSF, or hydroxyalkyl
starches, in particular with hydroxyethyl starch. The G-CSF can be
glycosylated or non-glycosylated. While the recombinant protein
that is produced in E. coli has no carbohydrate structures and is
expressed with an N-terminal methionine residue, the G-CSF that is
produced in eukaryotic cells, like for example CHO cells, is
normally glycosylated.
[0038] Preferably, the G-CSF contained in the liquid formulation is
human Met-G-CSF, produced in E. coli cells. Various expression
systems are commercially available for the expression in E. coli
cells. Suitable, for example, is the expression of human G-CSF
under the control of an inducible promoter, for instance an
IPTG-inducible promoter. See, for example, Sambrook and Russel,
Molecular Cloning--A Laboratory Manual, 3.sup.rd edition 2001, Cold
Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., chapter
15, or established manufacturers' protocols, for example by Promega
or Stratagene.
[0039] The fermentation of the host cells can be conducted
according to standard protocols, like they are described in patent
and scientific literature, as can the subsequent purification
including harvesting the so-called inclusion bodies, which contain
the G-CSF that is overexpressed in E. coli, lysing, solubilizing,
refolding, and chromatographically purifying said inclusion bodies;
suitable protocols can be found both in patent literature and in
standard references of protein chemistry as well as in laboratory
manuals.
[0040] The isolation and purification of G-CSF, including
solubilizing and refolding, are described, for example, in EP-A-0
719 860. General techniques for solubilizing and renaturing
denatured proteins have been described in EP-A-0 512 097, EP-A-0
364 926, EP-A-0 219 874, and WO 01/87925 and can moreover be taken
from scientific literature and standard references of protein
chemistry.
[0041] The refolded protein is subsequently purified by means of
chromatographic methods, i.e. it is separated from other proteins
and further contaminations that are contained after solubilizing
and renaturing.
[0042] Among others, patent document WO 87/01132 A1, which was
already referred to above and in which the production of G-CSF in
E. coli host cells was described for the first time, deals with
chromatographic purification. In the context of purifying the
recombinant G-CSF, a cation exchange chromatography using a CM
cellulose column is conducted in Example 7 in WO 87/01132 A1.
[0043] In EP 0 719 860 A1, the G-CSF is purified subsequently to
solubilization and oxidation, the solubilizer is removed by Dowex
treatment, followed by an anion exchange chromatography and a
cation exchange chromatography. In EP 0 719 860 A1, CM Sepharose is
also used for cation exchange chromatography.
[0044] In WO 03/051922 A1, a purification method for G-CSF is
described, in which a metal affinity chromatography is conducted,
more precisely an immobilized metal affinity chromatography (IMAC).
Subsequently to the metal affinity chromatography, a cation
exchange chromatography and/or a gel filtration can be conducted in
WO 03/051922.
[0045] In WO 01/04154 A1, a method for purifying G-CSF is
described, in which initially a hydrophobic interaction
chromatography and subsequently thereto a hydroxyapatite
chromatography are conducted. Subsequently to the hydroxyapatite
chromatography a cation exchange chromatography is performed.
[0046] The purity of the G-CSF that is formulated in the
formulation of the present invention should amount to at least 95%,
preferably to at least 97%, particularly preferably to at least 99%
and most preferably to more than 99%. Here, purity can be checked
by means of HPLC analyses. Suitable therefore are rp-SEC and IEX
analyses. The person skilled in the art can take suitable materials
and protocols for conducting the rpHPLC or the SEC-HPLC from
product information provided by suppliers like Vydac
(http://www.vydac.com) or TOSOH Bioscience
(http://www.tosohbiosep.de).
[0047] In the preparations according to the present invention,
G-CSF with a purity of more than 99%, preferably more than 99.5%,
is normally employed.
[0048] Preferably, the activity of the employed G-CSF should not be
lower than 50,000 IU/.mu.g; a G-CSF having an activity 80,000
IU/.mu.g is particularly suitable; most suitable is a G-CSF having
an activity of about 100,000 IU/.mu.g or more.
[0049] Determining the yield of Met-G-CSF is also described in
Herman et al. (1996) Pharm. Biotechnol. 9: 303-328. Here, the exact
ratio of Met-G-CSF is determined by means of integrating the peak
area and conversion on the basis of the extinction coefficient.
[0050] Subsequent to purification, the G-CSF can be analyzed with
respect to its amount and activity. A qualitative analysis can be
conducted via an SDS-PAGE followed by Coomassie Brilliant Blue
staining or via an rpHPLC. A commercially available G-CSF
preparation can be used as a standard for the analyses. In
addition, a peptide map or a mass spectroscopy can be performed.
The activity of the purified G-CSF can be determined by means of
different biological test methods, like they are described, for
example, in Shirafuji et al. (1989) Exp. Hematol. 17(2): 116-119;
Oh-Eda et al. (1990) J. Biol. Chem. 265(20): 11432-11435; Stute et
al. (1992) Blood 79(11): 2849-2854 and Oshima et al. (2000)
Biochem. Biophys. Res. Commun. 267(3): 924-927.
[0051] Incidentally, all the chromatographies are conducted
according to the recommendations and protocols of the suppliers of
the matrices or columns (for example with respect to flow rate,
column volumes employed for washing or for elution, diameters and
bed heights of the columns, etc.).
[0052] The biological activity of the obtained recombinant G-CSF
can be determined by means of a bioassay and can be compared to the
biological activity of a standard, i.e. of commercially available
G-CSF (Neupogen.RTM.). To this end, the murine cell line NFS-60,
which is responsive to G-CSF, can be used. For this purpose, said
cell line is cultivated in RPMI 1640 medium (Bachem, Heidelberg,
Germany) that contains 1.5 g/l sodium carbonate, 4.5 g/l glucose,
10 mM Hepes and 1.0 mM sodium pyruvate and is supplemented with 2
mM glutamine, 10% FCS, 0.05 mM 2-mercaptoethanol and 60 ng/ml
G-CSF.
[0053] For the activity test, the cells are washed twice with
G-CSF-free medium, seeded in 96-well plates at a concentration of
2.times.10.sup.4 cells per well and incubated for three days at
37.degree. C. and 4.5% CO.sub.2 with different concentrations of
the purified G-CSF and of the standard. Subsequently, the cells are
stained with XTT Reagent and absorption is measured at 450 nm in a
microtiter plate reader. It is desired that the cells treated with
the obtained G-CSF grow as well as the cells treated with the
standard (for example the trade product Neupogen.RTM.), as in this
case an equal biological activity of the two G-CSF samples can be
assumed.
[0054] The production of the formulation is also conducted
according to methods conventional in the art.
[0055] Conventionally, the buffer and tenside components and,
optionally, further pharmaceutically acceptable excipients are
first dissolved at suitable amounts in the aqueous solvent,
normally in sterile water. If necessary, the pH value is adjusted
by means of acetate solution or with other acids or bases, like the
exemplary ones mentioned above. Subsequently to a conventional
sterilization step, for example filtration through a sterile
filter, G-CSF is added at the desired concentration. However, it is
also easily possible to provide G-CSF in an aqueous solution and to
subsequently adjust the pH with acetate and/or suitable acids or
bases, preferably NaOH, to the desired value.
[0056] Eventually, the prepared liquid formulation is filled into a
suitable receptacle, where it is stored until application. In
particular, the receptacle is a prefilled syringe, a vial or an
ampoule.
[0057] The compositions according to the present invention can be
employed in the various forms of administration. The formulations
according to the present invention are, for example, suitable as
injection or infusion solutions, in particular for intravenous,
intramuscular, or subcutaneous administration. However, the
compositions can also be used for producing other forms of
administration, for example transfersomes, liposomes or
hydrogels.
[0058] The liquid formulation according to the present invention
does not only provide the advantages of omitting potentially
immunogenic compounds and altogether containing a minimum possible
number of ingredients, it also does not require lyophilization in
any phase of the production process. Thus, the expenses related to
lyophilization are saved on the one hand, and the risks of
mechanical problems, for example if the lyophilisate cannot be
reconstituted in a complete or sufficient manner, are avoided on
the other hand.
[0059] Within the scope of the present invention, the term
"storage-stable" is understood to denote that the content of active
G-CSF molecules is still 80% or more of the initial concentration
after storing the G-CSF liquid formulation for three months at
25.degree. C. Preferably, the residual content of G-CSF activity is
still at least 85%, more preferably at least 90%, and most
preferably at least 95% of the initial activity after three months
of storage at 25.degree. C. The activity of the G-CSF can be
determined by means of conventional activity tests, like already
described for G-CSF in the art. Within the scope of the present
invention, the term "liquid formulation" is understood to denote
that the formulation of G-CSF, together with further substances
contained in the formulation, is not lyophilized in any phase of
the production process, i.e. neither before nor during nor
subsequently to mixing the substances, and that the formulation is
intended for intravenous or subcutaneous application as injection
solution or infusion solution.
[0060] In a preferred embodiment, the formulation contains no
further ingredients other than the agent G-CSF, a tenside selected
from polysorbate 20, polysorbate 80 or a mixture thereof, acetate,
sorbitol, sodium ions, and water and has a pH value in the range of
between 4.2 to 4.3 or in the range of between 4.25 to 4.35, in
particular in the range of between 4.25 to 4.3. The use of
polysorbate 20 as the only tenside in the formulation is
particularly preferred.
EXAMPLES
[0061] The following Examples are employed to illustrate the
invention without limiting it thereby.
[0062] Compositions containing G-CSF were prepared by first
dissolving the buffer substance acetate in form of the sodium salt
together with polysorbate 20 or polysorbate 80 and sorbitol in
distilled and sterile water and subsequently adjusting the pH value
with NaOH to the desired value between 4.2 and 4.3.
Non-glycosylated recombinant human G-CSF (Filgrastim, Met-G-CSF)
was added at the desired concentration. Preparing and filling the
formulation in prefilled syringes was preferably conducted in a
nitrogen atmosphere.
[0063] The specific formulations of the compositions prepared
according to the present invention as well as their pH values are
given in the following tables. The use of polysorbate 80 instead of
polysorbate 20 led to comparable results. The same applies to the
use of mannitol instead of sorbitol.
TABLE-US-00001 Ingredient Formul. 1 Formul. 2 Formul. 3 Formul. 4
Formul. 5 Formul. 6 G-CSF 0.6 0.6 0.6 0.96 0.96 0.96 mg/ml Sorbitol
5.0 5.0 5.0 5.0 5.0 5.0 %, w/v Acetate buffer 10 10 10 10 10 10 mM
Polysorbate 20 0.004 0.004 0.004 0.004 0.004 0.004 %, w/v NaOH 0.1
n* q.s. q.s. q.s. q.s. q.s. q.s. Water 1.0 ml ad 1.0 ml ad 1.0 ml
ad 1.0 ml ad 1.0 ml ad 1.0 ml pH 4.2 4.25 4.3 4.2 4.25 4.3 *for
adjusting the pH value
[0064] The formulations according to the present invention were
stored at different temperatures for different periods of time
together with corresponding formulations having a pH value of 4.0,
which represent the state of the art and served as comparison
formulations.
[0065] Some data from long-term stability analyses are presented in
the appended Figures (G-CSF concentration 0.6 mg/ml in each case).
FIG. 1 shows SEC analyses and FIG. 2 shows IEX analyses, in each
case with a storage time of up to 6 months at 30.degree. C. and
40.degree. C., respectively.
[0066] All in all, the formulations according to the present
invention showed results comparable to those of the comparison
formulation, which only differed from the tested formulations
according to the present invention by exhibiting a more acidic pH
value, i.e. pH 4.0.
* * * * *
References